Synthesis of the amide analog of alternariolide (AM-toxin I), a host specific phytotoxin for apple leaves

Article abstract of DOI:10.3987/com-02-s63

The amide analog of alternariolide (AM-toxin I) was synthesized with high efficiency. The analog showed comparable biological activity to that of alternariolide.

Full text of DOI:10.3987/com-02-s63

HETEROCYCLES, Vol. 59, No. 2, 2003, pp. 623 - 633, Received, 26th August, 2002
SYNTHESIS OFTHEAMIDE ANALOG OFALTERNARIOLIDE
(AM-TOXIN I), A HOST SPECIFIC PHYTOTOXIN FORAPPLE LEAVES†
Mitsuru Sakai, Toshikatsu Okuno, Kimiko Hashimoto*2, and Haruhisa Shirahama*1
School of Science, Kwansei Gakuin University, Uegahara, Nishinomiya 662-8501, Japan
Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science,
Hirosaki University, Hirosaki 036-8561, Japan
e-mail (H. S.): shiraham@sci.hokudai.ac.jp; (K. H.) kimikoh@postman.riken.go.jp
Abstract - The amide analog of alternariolide (AM-toxin I) was synthesized with high
efficiency. The analog showed comparable biological activity to that of alternariolide.
Alternariolide (AM-toxin I, 1) produced by Alternaria mali has been found to be responsible for the necrotic brown
spots on certain apple leaves, which is the first example of a host-specific phytotoxin.1 The fungal pathogen,
Alternaria mali, produces alternariolide (1) and its congeners, AM-toxin II (2) and III (3), in the infection processes to
the host plants. These compounds have been considered to function as host recognition factors.2
X
OMe
CO
CO
L-Amp
HN
CONH
∆ Ala
COO
L-Ala
CONH
L-Hmb
HN
CONH
∆ Ala
CONH
CONH
L-Ala L-Val
4
Alternariolide (AM-toxin I, 1) : X = OMe
AM-toxin II (2) : X = H
AM-toxin III (3) : X = OH
† Dedicated to Professor Yuichi Kanaoka in celebration of his 75th birthday.

Scheme 1
OMe
SePh
H N
2
CO H
2
BocHN
CO H
BocHN
CO H
HCl•H N
CO Bzl
2
2
2
2
H
H
H
H
L-Amp (9)
Boc-D-PhSeAla (15)
Boc-L-Ala (6)
HCl•L-Val-OBzl (5)
Boc O, Et N
2
3
/ dioxane - H O quant.
2
Boc
OH
91%
t
10
t
O Bu
i
i
/ CH Cl
NPr
PrHN
2
2
92%
OBzl
DCC, HOBT, NMM / THF
Boc
Boc
Boc
OBu 11
7
3M HCl / AcOEt
HCl •H
77%
H , 10% Pd-C / MeOH
2
89%
OH
t
OBu
12
13
14
16
8
DCC, HOBt, NMM / THF
97%
t
Boc
OBu
0.89M HCl / AcOEt
EDC, HOBt / DMF
87%
t
HCl•H
OBu
95%
SePh
Boc
t
OBu
1) TFA 2) 3 M HCl / dioxane
quant.
SePh
HCl•H
OH 17
i
FDPP, Pr NEt / 0.5 mM DMF
SePh
2
77%
84%
cyclo(
)
)
18
4
TBHP / CH Cl - TFE (3 : 1)
2
2
cyclo( ∆ Ala
L-Amp : L-2-Amino-5-(4-methoxyphenyl)pentanoic acid
HOBt : 1-Hydroxybenzotriazole
NMM : N-Methylmorpholine
TFE : 2,2,2-Trifluoroethanol
FDPP : Pentafluorophenyl diphenylphosphinate
Accordingly, these phytotoxins should be key compounds in revealing the mechanism of the host-specificity.
Recently, we developed a new synthetic method for 1 with high efficiency.1g To examine the applicability of the
method for other derivatives,3 we planned the synthesis of the amide analog (4).4 One of the structural properties of
alternariolide (1) is the relatively rigid cyclic structure composed of three amide bonds, one ester bond, and one

dehydroamino acid residue. Changing the ester bond to an amide bond should make its conformation more rigid
because of the planarity of the amide bond. Accordingly, the cyclization step is estimated to be harder than that of 1.
Izumiya has previously synthesized the same peptide analog through a similar intermediate.4a During the synthesis, he
encountered a difficulty in the cyclization step. The intermediate for the peptide analog resists the cyclization
compared to the ester analog.
Additionally, the conformation of the alternariolide derivative might be an important factor in the biological activity,
because the analogs composed of the reduced form (both L- and D-forms) of the dehydroalanine show weak activities.5
HCl•L-Val-OBzl (5) was synthesized from L-Val as follows; 1) protection of the amine with a Boc group, 2)
esterification of the carboxylic acid with benzyl chloride, 3) deprotection of the Boc group under acidic conditions.
The HCl salt (5) was condensed with Boc-L-Ala (6) using DCC, HOBT, and NMM to give peptide (7) in 92% yield.
Hydrogenolysis of the benzyl ester of the peptide (7) gave carboxylic acid (8) in 89% yield. A non-proteinogenic
amino acid, L-Amp (9), was synthesized according to Shimohigashi’s report6 with slight modifications. For the
condensation of L-Amp (9) with the peptide (8), the protecting groups of 9 were arranged as follows; 1) protection of
the amino group as the Boc carbamate giving 10, 2) esterification of the carboxylic acid using t-butylisourea7 giving t-
butyl ester (11), and 3) deprotection of the Boc group keeping the t-butyl ester intact under acidic conditions,8 affording
the HCl salt (12). The thus prepared HCl salt (12) was combined with 8 to afford tripeptide (13) in 97% yield. After
removal of the Boc group of 13, the resulting HCl salt (14) was condensed with Boc-D-PhSeAla9 (15) to give
tetrapeptide (16). The protecting groups of both ends in 16 were removed with TFA, and the resulting TFA salt was
changed to a stable HCl salt (17). Cyclization of 17 was performed at a concentration of 0.5 mM in DMF with
FDPP10 to give cyclic peptide (18) in 77% yield. In our synthesis of alternariolide (1), cyclization succeeded at a 4
mM concentration; however, this peptide derivative (17) produced its dimer as the main product at the same
concentration. At the last stage, the phenylselenyl group was oxidatively eliminated from 18 to produce the peptide
analog (4) in 84% yield. Thus our synthetic method has been found to be applicable to the more rigid amide derivative.
In this synthesis, the key steps are the cyclization and the dehydroalanine formation steps. The yields for these steps in
the previous synthesis by the Izumiya group are 19% and 27%, respectively.4a, 11 On the other hand, our process has
been efficiently improved, affording 77% and 84% yields, respectively. The biological activity of 4 on susceptible

apple leaves was comparable to that of alternariolide (1), of which the least effective dose was 0.001 µg / mL in our
bioassay system.1h The 1H NMR spectrum of 4 appears to be more complex than that of 1,5 which might be due to the
presence of some conformers.4a However, the conformational change did not affect the biological activity.
EXPERIMENTAL
HCl•L-Val-OBzl (5)
To a solution of L-Val (10.0 g, 85.5 mmol) in H2O (100 mL) were added Et3N (13.1 mL, 94.1 mmol) and a solution of
Boc2O (22.4 g, 103 mmol) in dioxane (100 mL) and the solution was stirred at rt overnight. The solution was
concentrated to about half of the volume in vacuo and adjusted to pH 2 with 1M HCl. The mixture was extracted with
EtOAc (100 mL x 3). The combined organic phase was washed with brine and dried over anhydrous Na2SO4. The
filtered solution was concentrated in vacuo to give Boc-L-Val-OH as a colorless oil (18.5 g, quant). The crude product
was used for the next step without further purification. [α]D -5.3° (c 0.94, CH3CO2H) [lit.,13 5.8° 1.208%, AcOH; mp
77-79 °C]; 1H NMR (400 MHz, CDCl3, TMS = 0 ppm) δ 0.93 (3H, d, J = 6.6 Hz), 1.00 (3H, d, J = 6.6 Hz), 1.45 (9H, s),
2.19 (1H, m), 4.04 (ca. 1/4H, br), 4.24 (ca. 3/4H, m), 5.08 (ca. 7/10H, d, J = 8.5 Hz), 6.14 (ca. 3/10H, br s), 8.40 (1H, br
s); IR (cm-1, neat) 3326, 2973, 1688, 1512, 1370, 1169, 1094, 1019, 862, 777.
The crude Boc-L-Val-OH (16.0 g, 73.6 mmol) was dissolved in DMF (160 mL). To the solution were added Et3N
(41.2 mL, 294 mmol) and BzlCl (25.4 mL, 221 mmol) at 0 °C and the mixture was stirred overnight at rt overnight.
The resulting solid (Et3N•HCl) was filtered off. After addition of H2O to the solution, the mixture was extracted with
EtOAc (150 mL x 3). The combined organic phase was successively washed with 1M HCl (150 mL x 3), H2O (150
mL x 3), and brine and dried over anhydrous Na2SO4. The filtered solution was concentrated in vacuo and the residue
was chromatographed on silica gel (20% EtOAc-hexane) to give ester (19.2 g, 85%) as a colorless oil. [α]D -2.7° (c
1.03, CHCl3) [lit.,14 -33.3°, c 2, MeOH]; 1H NMR (400 MHz, CDCl3, TMS = 0 ppm) δ 0.85 (3H, d, J = 6.8 Hz), 0.94
(3H, d, J = 6.8 Hz), 1.44 (9H, s), 2.15 (1H, m), 4.27 (1H, dd, J = 4.6, 8.6 Hz), 5.13 (1H, d, J = 12.4 Hz), 5.21 (1H, d, J =
12.4 Hz), 7.35 (5H, s); IR (cm-1, neat) 3378, 2971, 1717, 1505, 1368, 1248, 1157, 1090, 1015, 750.
Boc-L-Val-OBzl (21.5 g, 69.9 mmol) was dissolved in TFA (200 mL) and the solution was stirred for 1 h at rt. After
evaporation of TFA, the residue was dissolved in a solution of 3M HCl in dioxane (46 mL, 138 mmol) and to the

mixture was added Et2O for crystallization. The resulting colorless needles were collected to give 5 (14.4 g, 85%).
[α]D -4.6° (c 1.01, MeOH) [lit.,15 -10.6° (c 2, 1N HCl), lit.,16 9.1° (c 3, pyridine)]; mp 138 °C (recryst. from Et2O -
MeOH) [lit.,15 142-143 °C, lit.,16 138-139 °C]; 1H NMR (400 MHz, D2O, HOD = 4.65 ppm) δ 0.82 (3H, d, J = 8.2 Hz),
0.84 (3H, d, J = 8.2 Hz), 2.20 (1H, m), 3.90 (1H, d, J = 4.6 Hz), 5.13 (1H, d, J = 12.0 Hz), 5.20 (1H, d, J = 12.0 Hz),
7.31 (5H, m); IR (cm-1, KBr disk) 2971, 2863, 1742, 1586, 1503, 1223, 1105, 1042, 741, 698.
Boc-L-Ala-L-Val-OBzl (7)
To a suspension of 5 (10.0 g, 41.0 mmol) in THF (250 mL) were added N-methylmorpholine (4.51 mL, 41.0 mmol),
Boc-L-Ala-OH (6, 7.76 g, 41.0 mmol), HOBT (6.10 g, 45.1 mmol), and DCC (9.31 g, 45.1 mmol) at 0 °C. The
mixture was stirred for 30 min at 0 °C and at rt overnight. The resulting precipitates were filtered off and the mother
liquor was concentrated to dryness. To the residue was added a small amount of EtOAc and the resulting insoluble
urea was again filtered off. The solution was concentrated in vacuo and the residue was chromatographed on silica gel
(20% EtOAc-hexane) to give 7 (14.2 g, 92%) as a colorless oil. [α]D -36.5° (c 0.92, CHCl3); 1H NMR (400 MHz,
CDCl3, TMS = 0 ppm) δ 0.86 (3H, d, J = 6.8 Hz), 0.91 (3H, d, J = 6.8 Hz), 1.34 (3H, d, J = 7.1 Hz), 1.44 (9H, s), 2.20
(1H, m), 4.19 (1H, m), 4.58 (1H, dd, J = 4.9, 8.0 Hz), 5.03 (1H, d, J = 4.9 Hz), 5.13 (1H, d, J = 12.2 Hz), 5.20 (1H, d, J
= 12.2 Hz), 6.70 (1H, d, J = 8.0 Hz), 7.35 (5H, m); IR (cm-1, neat) 3322, 2973, 1738, 1667, 1522, 1368, 1250, 1171, 752,
698; EI-HR-MS 378.2153 [M]+ calcd for C20H30N2 O5 378.2154.
Boc-L-Ala-L-Val-OH (8)
In a round-bottomed flask was placed 10% Pd/C (3.9 g). After addition of small amount of H2O, air in the flask was
replaced with argon. To the flask was added 7 (14.0 g, 37.0 mmol) in MeOH (140 mL). After replacement of the
argon with H2, the mixture was stirred at rt overnight. The mixture was carefully filtered through a pad of Celite with
suction. The resulting solution was evaporated in vacuo to give crude carboxylic acid (8) (9.49 g, 89%) as a colorless
amorphous powder. [α]D +8.6° (c 1.00, CHCl3); mp 155 °C; 1H NMR (400 MHz, CDCl3, TMS = 0 ppm) δ 0.94 (3H,
d, J = 6.7 Hz), 0.97 (3H, d, J = 6.7 Hz), 1.35 (3H, d, J = 7.1 Hz), 1.44 (9H, s), 2.25 (1H, m), 4.26 (1H, m), 4.56 (1H, dd,
J = 4.9, 9.1 Hz), 5.23 (1H, br s), 6.96 (1H, d, J = 9.1 Hz); IR (cm-1, KBr disk) 3364, 2978, 1707, 1634, 1553, 1507, 1252,

1171, 1073, 1024; EI-HR-MS 288.1686 [M]+ calcd for C13H24N2 O5 288.1685.
Boc-L-Amp-OH (10)
L-Amp (9) was prepared according to Shimohigashi’s report with slight modification. To a solution of L-Amp (9,
5.17 g, 23.2 mmol) in H2O (52 mL) were added Et3N (3.56 mL, 25.5 mmol) and a solution of Boc2O (6.06 g, 27.8
mmol) in dioxane (52 mL) and the solution was stirred at rt overnight. The solution was concentrated to about half of
the volume in vacuo and adjusted to pH 2 with 1M HCl. The mixture was extracted with EtOAc (50 mL x 3). The
combined organic phase was washed with brine and dried over anhydrous Na2SO4. The filtered solution was
concentrated in vacuo to give Boc-L-Amp-OH (10) as a colorless oil (7.48 g, quant). The crude product was used for
the next step without further purification. [α]D +5.9° (c 0.88, MeOH); 1H NMR (400 MHz, CDCl3, TMS = 0 ppm) δ
1.42 (9H, s), 1.66 (3H, m), 1.85 (1H, m), 2.55 (2H, m), 3.77 (3H, s), 4.28 (1H, m), 5.11 (1H, br s), 5.38 (1H, br s), 6.80
(2H, d, J = 8.3 Hz), 7.06 (2H, d, J = 8.3 Hz); IR (cm-1, neat) 3343, 2978, 2936, 1713, 1512, 1368, 1246, 1173, 1036,
831; EI-HR-MS 323.1751 [M]+ calcd for C17H25N O5 323.1733.
Boc-L-Amp-OtBu (11)
To a solution of 12 (4.4 g, 13.6 mmol ) in CH2Cl2 (30 mL) was added O-t-butyl-N,N’-diisopropylisourea (4.08 g, 20.4
mmol) at rt and the solution was heated under reflux overnight. To the mixture was added additional amount of O-t-
butyl-N,N’-diisopropylisourea (3.81 g, 19.0 mmol) and the mixture was refluxed for 7 h. After cooling to rt, the
resulting insoluble urea was filtered off and the mother liquor was concentrated in vacuo. To the residue was added a
mixture of EtOAc and hexane (1:9) and the insoluble material was again removed. The solution was concentrated and
the residue was chromatographed on silica gel (10% EtOAc-hexane) to afford ester (13) (4.69 g, 91%) as a colorless oil.
[α]D +11.6° (c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3, TMS = 0 ppm) δ 1.44 (18H, s), 1.68 (4H, m), 2.57 (2H, m),
3.78 (3H, s), 4.20 (1H, m), 5.00 (1H, d, J = 8.1 Hz), 6.82 (2H, d, J = 8.5 Hz), 7.07 (2H, d, J = 8.5 Hz); IR (cm-1, neat)
2978, 2934, 1717, 1512, 1458, 1368, 1248, 1155, 1038, 845; EI-HR-MS 379.2358 [M]+ calcd for C21H33NO5 379.2359.
HCl•L-Amp-OtBu (12)
Boc-L-Amp-OtBu (11, 4.74 g, 12.5 mmol) was dissolved in 0.89 M HCl / EtOAc (70 mL, 62.3 mmol) and the solution

was stirred for 5h at rt. The solution was concentrated in vacuo and the residue was washed with Et2O to afford HCl
salt (12, 3.04 g, 77%) as colorless crystals. [α]D +12.3° (c 0.52, MeOH); mp 143 °C (recryst from MeOH-Et2O); 1H
NMR (400 MHz, D2O, HOD = 4.65 ppm) δ 1.32 (9H, s), 1.49 (1H, m), 1.64 (1H, m), 1.77 (2H, m), 2.52 (2H, m), 3.69
(3H, s), 3.86 (1H, t, J = 5.3 Hz), 6.85 (2H, d, J = 8.8 Hz), 7.11 (2H, d, J = 8.8 Hz); IR (cm-1, nujol) 2924, 2855, 1732,
1510, 1244, 1157; FAB-HR-MS 280.1888 [M+H]+ calcd for C16H26N O3 280.1913.
t
Boc-L-Ala-L-Val-L-Amp-OBu (13)
To a suspension of 12 (675 mg, 2.14 mmol) in THF (15 mL) were added N-methylmorpholine (0.24 mL, 2.14 mmol),
dipeptide (8, 616 mg, 2.14 mmol), and HOBt (318 mg, 2.35 mmol) and the solution was cooled to 0°C. To the
solution was added DCC (485 mg, 2.35 mmol) and the mixture was stirred for 30 min at 0°C and then at rt overnight.
The resulting insoluble urea was filtered off and the mother liquor was concentrated to dryness. The residue was
chromatographed on silica gel (1% EtOAc-CHCl3) to give tripeptide (13, 1.14 g, 97%) as colorless crystals. [α]D -
34.0° (c 1.01, CHCl3); mp 130-131 °C (recryst. from CHCl3-pet ether); 1H NMR (400 MHz, CDCl3, TMS = 0 ppm) δ
0.92 (3H, d, J = 6.8 Hz), 0.94 (3H, d, J = 6.8 Hz), 1.32 (3H, d, J = 7.1 Hz), 1.43 (18H, s), 1.59 (2H, m), 1.83 (2H, m),
2.15 (1H, m), 2.54 (2H, m), 3.78 (3H, s), 4.18 (1H, br s), 4.28 (1H, m), 4.48 (1H, dd, J = 6.1, 13.4 Hz), 5.11 (1H, br s),
6.55 (1H, br s), 6.81 (3H, m), 7.05 (2H, d, J = 8.5 Hz); IR (cm-1, KBr disk) 3318, 2976, 1642, 1514, 1456, 1248, 1040,
845, 700, 652; EI-HR-MS 549.3416 [M]+ calcd for C29H47N3O7 549.3414.
HCl•L-Ala-L-Val-L-Amp-OtBu (14)
Tripeptide (13, 4.5 g, 8.19 mmol) was dissolved in 0.89 M HCl / EtOAc (46 mL, 40.9 mmol) and the mixture was
stirred for 5h at rt. When the solution became turbid, a small amount of i-PrOH was added. The solution was
concentrated in vacuo and the residue was washed with Et2O to afford HCl salt (14, 3.48 g, 87%) as a colorless
amorphous powder. [α]D -22.0° (c 0.30, MeOH); mp 179-180 °C (recryst .from MeOH-Et2O); 1H NMR (400 MHz,
DMSO-d6, DMSO = 2.49 ppm) δ 0.86 (3H, d, J = 6.8 Hz), 0.89 (3H, d, J = 6.8 Hz), 1.29 (3H, d, J = 6.8 Hz), 1.35 (9H,
s), 1.59 (4H, m), 1.98 (1H, m), 2.49 (2H, m), 3.70 (3H, s), 3.93 (1H, m), 4.07 (1H, m), 4.24 (1H, m), 6.82 (2H, d, J = 8.5
Hz), 7.06 (2H, d, J = 8.5 Hz), 8.24 (3H, br s), 8.31 (1H, d, J = 7.1 Hz), 8.46 (1H, m); IR (cm-1, nujol) 3287, 2924, 2855,

1736, 1651, 1512, 1248, 1155; FAB-HR-MS 450.2984 [M+H]+ calcd for C24H40N3 O5 450.2968.
t
Boc-D-PhSeAla-L-Ala-L-Val-L-Amp-OBu (16)
To a solution of Boc-D-PhSeAla (15, 2.41 g, 7.00 mmol) in DMF (60 mL) were added tripeptide (14, 3.40 g, 7.00
mmol) and HOBT (1.04 g, 7.70 mmol). To the cooled solution at -5 °C was added EDC (1.34 mL, 7.35 mmol). The
solution was degassed under reduced pressure and filled with argon. After stirring 30 min at -5 °C, the solution was
stirred at rt overnight. To the solution was added cooled H2O and the resulting precipitate was collected. The crude
crystals were recrystallized from EtOAc-hexane to give tetrapeptide (16, 5.42 g, 95%). [α]D +12.9° (c 0.75, DMF);
mp 199 °C (recryst .from CHCl3-hexane); 1H NMR (400 MHz, DMSO-d6, DMSO = 2.49 ppm) δ 0.82 (3H, d, J = 6.6
Hz), 0.85 (3H, d, J = 6.6 Hz), 1.13 (3H, d, J = 7.1 Hz), 1.35 (9H, s), 1.36 (9H, s), 1.58 (4H, m), 1.95 (1H, m), 2.47 (2H,
m), 3.06 (1H, m), 3.22 (1H, m), 3.69 (3H, s), 4.10 (1H, m), 4.18 (2H, m), 4.31 (1H, m), 6.81 (2H, d, J = 8.4 Hz), 7.02
(1H, d, J = 9.0 Hz), 7.06 (2H, d, J = 8.4 Hz), 7.27 (3H, m), 7.48 (2H, m), 7.75 (1H, d, J = 9.5 Hz), 8.03 (1H, d, J = 7.8
Hz), 8.15 (1H, d, J = 7.3 Hz) ; IR (cm-1, nujol) 3295, 2924, 2855, 1711, 1634, 1512, 1246, 1163; FAB-HR-MS
777.3357 [M+H]+ calcd for C38H57N4 O880Se 777.3342.
HCl•D-PhSeAla-L-Ala-L-Val-L-Amp-OH (17)
Tetrapeptide (16, 3.86 g, 4.98 mmol) was dissolved in TFA (38 mL) and the solution was stirred for 2 h at rt. After
evaporation, the residue was dissolved in 3M HCl / dioxane (3.5 mL, 10.5 mmol). To the solution was added Et2O
and the resulting precipitate was collected to afford HCl salt (17, 3.26 g, quant) as a colorless amorphous powder. [α]D
-35.9° (c 0.85, DMF); mp 134-135 °C (recryst. from MeOH-Et2O); 1H NMR (400 MHz, DMSO-d6, DMSO = 2.49
ppm) δ 0.83 (3H, d, J = 6.7 Hz), 0.85 (3H, d, J = 6.7 Hz), 1.06 (3H, d, J = 6.8 Hz), 1.61 (4H, m), 1.96 (1H, m), 2.46 (2H,
m), 3.34 (2H, m), 3.69 (3H, s), 4.08 (1H, m), 4.17 (2H, m), 4.33 (1H, m), 6.81 (2H, d, J = 8.6 Hz), 7.06 (2H, d, J = 8.6
Hz), 7.30 (3H, m), 7.53 (2H, m), 8.07 (1H, d, J = 9.2 Hz), 8.14 (1H, d, J = 7.6 Hz), 8.72 (1H, d, J = 7.2 Hz) ; IR (cm-1,
nujol) 3272, 2924, 2855, 1640, 1545, 1512, 1246, 1177; FAB-HR-MS 621.2211 [M+H]+ calcd for C29H41N4 O680Se
621.2191.
Cyclo(D-PhSeAla-L-Ala-L-Val-L-Amp) (18)

HCl salt (17, 400 mg, 0.610 mmol) was dissolved in DMF (1.2 mL). To the solution were added i-Pr2NEt (0.44 mL,
2.50 mmol) and FDPP (472 mg, 1.22 mmol). The solution was degassed under reduced pressure and filled with argon.
After stirring for 5 h at rt, the solution was concentrated in vacuo. The residue was dissolved in CHCl3-TFE and the
solution was successively washed with 1M HCl (10 mL x 1), sat. NaHCO3 (10 mL x 1), and brine. The solution was
dried over anhydrous Na2SO4 and evaporated in vacuo. The residue was washed with Et2O and recrystallized from
CHCl3-TFE-Et2O to afford cyclic peptide (18, 284 mg, 77%) as colorless crystals. [α]D -102.8° (c 0.13, DMF); mp
309-310 °C (decomp); 1H NMR (400 MHz, DMF-d7, DMF-CHO = 8.01 ppm) δ 0.89 (6H, m), 1.34 (3H, d, J = 7.3 Hz),
1.65 (4H, m), 2.10 (1H, m), 2.55 (2H, m), 3.07 (1H, m), 3.28 (1H, m), 3.76 (3H, s), 3.81 (1H, d, J = 9.3 Hz), 4.33 (2H,
m), 4.62 (1H, m), 6.85 (2H, d, J = 7.8 Hz), 7.12 (2H, d, J = 7.8 Hz), 7.29 (3H, m), 7.54 (2H, m), 7.63 (1H, d, J = 9.3 Hz),
7.86 (1H, d, J = 9.8 Hz), 8.00 (2H, m) ; IR (cm-1, nujol) 3285, 2924, 2855, 1651, 1536, 1512, 1246, 1036; EI-HR-MS
602.2030 [M]+ calcd for C29H38N4 O580Se 602.2007, 600.2006 [M]+ calcd for C29H38N4 O578Se 602.2016.
∆
Cyclo( Ala-L-Ala-L-Val-L-Amp) (4)
To a solution of cyclic peptide (18, 39.8 mg, 0.0662 mmol) in CH2Cl2 (6 mL) and TFE (2 mL) was slowly added a
solution of TBHP in CH2Cl2 (4.03 M, 0.48 mL, 1.93 mmol) at -5 °C. After stirring for 6 h at rt, the mixture was
extracted with CHCl3 (10 mL x 3). The combined extracts were washed with sat. NaHCO3 (10 mL x 3) and brine.
The organic phase was dried over anhydrous Na2SO4 and evaporated in vacuo. The residue was washed with Et2O
and chromatographed on silica gel (5% i-PrOH-CHCl3) to give dehydropeptide (4, 24.7 mg, 84%) as colorless crystals.
[α]D -176.1° (c 0.055, DMF); mp 199-200 °C (decomp, recryst from CHCl3-hexane)[lit.,4a mp 220-223 °C]; 1H NMR
(400 MHz, DMF-d7, DMF-CHO = 8.01 ppm, -20 °C) δ 0.88 (6H, m), 1.13 (3/2H, d, J = 6.0 Hz), 1.38 (3/2H, d, J = 6.9
Hz), 1.35-2.00 (9/2H, m), 2.15 (1/2H, m), 2.49 (2H, m), 3.53 (1/2H, m), 3.73, 3.74 (total 3H, s each), 3.89 (1/2H, m),
4.37 (1/2H, m), 4.69 (1/2H, m), 4.77 (1/2H, m), 4.86 (1/2H, m), 5.03 (1/2H, d, J = 8.3 Hz), 5.71 (1/2H, d, J = 20.0 Hz),
6.24 (1/2H, d, J = 20.0 Hz), 6.83 (2H, m), 7.10 (2H, m), 7.34 (1/2H, d, J = 8.3 Hz), 7.66 (1/2H, d, J = 10.4 Hz), 7.81
(1/2H, m), 8.20 (1/2H, d, J = 8.5 Hz), 8.61 (1/2H, d, J = 9.3 Hz), 8.71 (1/2H, m), 8.75 (1/2H, br s), 8.84 (1/2H, br s),
10.37 (1/2H, br s) ; IR (cm-1, nujol) 3281, 2924, 2855, 1686, 1642, 1512, 1246, 1036; FAB-HR-MS 445.2458 [M+H]+
calcd for C23H33N4 O5 445.2451.

REFERENCESAND NOTES
*1 Present address: 2-16, Minami 18 Nishi 8, Chuo-ku, Sapporo, 064-0918, Japan
*2 To whom correspondence should be addressed. Present address: Lab. of Biochemical Resources, Plant Science
Center, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan
1. Isolation: (a) T. Okuno, Y. Ishita, K. Sawai, and T. Matsumoto, Chem. Lett., 1974, 635. (b) T. Okuno, Y. Ishita, A.
Sugawara, Y. Mori, K. Sawai, and T. Matsumoto, Tetrahedron Lett., 1975, 335. (c) T. Ueno, T. Nakashima, Y.
Hayashi, and H. Fukami, Agric. Biol. Chem., 1975, 39, 1115. (d) idem, ibid., 1975, 39, 2081.
Synthesis of 1: (e) S. Lee, H. Aoyagi, Y. Shimohigashi, N. Izumiya, T. Ueno, and H. Fukami, Tetrahedron Lett.,
1976, 843. (f) Y. Shimohigashi, S. Lee, H. Aoyagi, T. Kato, and N. Izumiya, Int. J. Peptide Protein Res., 1977, 10,
323. (g) K. Hashimoto, M. Sakai, T. Okuno, and H. Shirahama, Chem. Commun., 1996, 1139.
Review: (h) K. Hashimoto, T. Okuno, M. Nakata, and H. Shirahama, ‘Current Topics in Phytochemistry,’ Vol. 3,
Research Trends, Trivandrum, India, 2000, pp. 1-37.
2. (a) J. D. Walton, Biochem. Pepti. Antibiotics, 1990, 179. (b) K. Kohmoto and H. Ohtani, Experientia, 1991, 47,
755.
3. (a) K. Hashimoto, H. Kawaguchi, M. Sakai, T. Okuno, and H. Shirahama, Synlett, 1997, 1202. (b) K. Hashimoto,
T. Yoshioka, C. Morita, M. Sakai, T. Okuno, and H. Shirahama, Chem. Lett., 1998, 203.
4. The amide derivative has been synthesized by Izumiya’s group by solution method and Miyashita’s group by solid
phase synthesis.: (a) H. Aoyagi, H. Mihara, T. Kozono, M. Waki, T. Kato, N. Izumiya, K. Ueda, and T. Ueno,
Peptide Chem., 1983, 53. (b) M. Miyashita, T. Nakamori, T. Murai, H. Miyagawa, M. Akamatsu, and T. Ueno,
Biosci. Biotechnol. Biochem., 2001, 62, 1799.
5. T. Higashijima, Y. Shimohigashi, T. Kato, N. Izumiya, T. Ueno, and T. Miyazawa, Biopolymers, 1983, 22, 1167.
6. Y. Shimohigashi, S. Lee, and N. Izumiya, Bull. Chem. Soc., Jpn, 1976, 49, 3280.
7. (a) V. E. Schmidt and F. Moosmueller, Liebigs Ann. Chem., 1955, 597, 235. (b) E. Schmidt and W. Carl, ibid.,
1961, 639, 24. (c) L. J. Mathias, Synthesis, 1979, 561.
8. F. S. Gibson, S. C. Bergmeier, and H. Rapoport, J. Org. Chem., 1994, 59, 3216

9. M. Sakai, K. Hashimoto, and H. Shirahama, Heterocycles, 1997, 44, 319.
10. S. Chen and J. Xu, Tetrahedron Lett., 1991, 32, 6711.
11. Miyashita reported the structure-activity relationship of the peptide analog in ref. 4b, but the synthetic scheme and
the yields were not clear.
12. Izumiya has reported that the necrotic activity of 4 was less active than that of 1. The effective doses were 0.1
mg/mL for 4 and 0.001 mg/mL for 1. Miyashita has also reported the activity using newly developed assay
-9
-10
method. The minimum concentrations for the necrosis were 10 for 4 and 10 M for 1. These differences
might be due to the difference in the bioassay systems.
13. G. W. Anderson andA. C. McGregor, J. Am. Chem. Soc., 1957, 79, 6180.
14. M. K. Dhaon, R. K. Olsen, and K. Ramasamy, J. Org. Chem., 1982, 47, 1962.
15. F. M. F. Chen and N. L. Benoiton, Int. J. Peptide Protein Res., 1986, 27, 221.
16. J. E. Shields, W. H. McGregor, and F. H. Carpenter, J. Org. Chem., 1961, 26, 1491.